Involvement of the Arg566 residue of Aeromonas sobria serine protease in substrate specificity

Abstract

Aeromonas sobria serine protease (ASP) is an extracellular serine protease secreted by the organism. Here, we identified the amino acid residue of ASP that contributes to substrate specificity by using both synthetic peptides and biological protein components. The results showed that the arginine residue at position 566 (Arg-566) of ASP, which is located in the extra occluding region of ASP close to an entrance of the catalytic cavity, is involved in the substrate specificity. A substitutional point mutation of the Arg-566 residue of ASP to Ala residue (ASP[R566A]) caused a decrease of the proteolytic efficiency for a certain substrate. In addition, ASP lost the ability to recognize the primary substrate by such a point mutation, and ASP[R566A] reacted to a wide range of synthetic substrates. It is likely that Arg-566 causes an interaction with the amino acid residue at position P3 of the substrate, which is the third amino acid residue upstream from the cleavage site. Another study using ORF2 protein, a chaperone protein of ASP, further suggested that Arg-566 could also play an important role in interaction with ORF2. We therefore conclude that the Arg-566 residue of ASP is likely responsible for the selection of substrates.

Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

“…In addition, a final concentration of 1 mM phenylmethylsulfonyl fluoride (PMSF) (Nacalai Tesque) was used as an ASP inhibitor. In our previous work, we confirmed that the ASP activity was completely inhibited by the presence of 1 mM PMSF [23].…”

“…Judging from the Km value, the affinity of ASP for the synthetic substrate Ala-Ala-Pro-Lys-pNA was not as high as that for the substrate Glu-Lys-Lys-MCA, but the Kcat / Km value corresponding to catalytic efficiency for substrate Ala-Ala-Pro-Lys-pNA was relatively high even though it can be inferred from our previous results [23], suggesting that a substrate with a PL sequence at its carboxyl terminus is a relatively good substrate for ASP.…”

“…Although the strength of the proteolytic activity against the synthetic substrate with a PL sequence at its carboxyl terminus (Ala-Ala-Pro-Lys-pNA) was slightly lower than that against the synthetic substrate Glu-Lys-Lys-MCA, which is a favorable substrate for ASP as reported [23], ASP markedly cleaved the synthetic peptide with a PL sequence at its carboxyl terminus but did not cleave another synthetic peptide with an AA sequence at its carboxyl terminus (Fig 6D). We also calculated the kinetic values (Table 1).…”

“…We measured the proteolytic activity of ASP as described [23]. Briefly, Boc-Glu-Lys-Lys-MCA, Glt-Ala-Ala-Pro-Leu- p NA, and Suc-Ala-Ala-Ala- p NA (Peptide Institute, Osaka, Japan) were used as synthetic peptide substrates for ASP.…”